Maria Sundvik

The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases.

Modulatory neurotransmitters which signal through G protein-coupled receptors control brain functions which deteriorate in degenerative brain diseases. During the past decade many of these systems have been mapped in the zebrafish brain. The main architecture of the systems in zebrafish brain resembles that of the mammals, despite differences in the development of the telencephalon and mesodiencephalon. Modulatory neurotransmitters systems which degenerate in human diseases include dopamine, noradrenaline, serotonin, histamine, acetylcholine and orexin/hypocretin. Although the number of G protein-coupled receptors in zebrafish is clearly larger than in mammals, many receptors have similar expression patterns, binding and signaling properties as in mammals. Distinct differences between mammals and zebrafish include duplication of the tyrosine hydroxylase gene in zebrafish, and presence of one instead of two monoamine oxidase genes. Zebrafish are sensitive to neurotoxins including MPTP, and exposure to this neurotoxin induces a decline in dopamine content and number of detectable tyrosine hydroxylase immunoreactive neurons in distinct nuclei. Sensitivity to important neurotoxins, many available genetic methods, rapid development and large-scale quantitative behavioral methods in addition to advanced quantitative anatomical methods render zebrafish an optimal organism for studies on disease mechanisms.

Modulatory Neurotransmitter Systems and Behavior: Towards Zebrafish Models of Neurodegenerative Diseases

The modulatory aminergic neurotransmitters are involved in practically all important physiological systems in the brain, and many of them are also involved in human central nervous system diseases, including Parkinsons disease, schizophrenia, Alzheimers disease, and depression. The zebrafish brain aminergic systems share many structural properties with the mammalian systems. The noradrenergic, serotonergic, and histaminergic systems are highly similar. The dopaminergic systems also show similarities with the major difference being the lack of dopaminergic neurons in zebrafish mesencephalon. Development of automated quantitative behavioral analysis methods for zebrafish and imaging systems of complete brain neurotransmitter networks have enabled comprehensive studies on these systems in normal and pathological conditions. It is possible to visualize complete neurotransmitter systems in the whole zebrafish brain at an age when the fish already displays all major vital behaviors except reproduction. Alterations of brain dopaminergic systems with MPTP, the neurotoxin that in humans and rodents induces Parkinsons disease, induces both changes in zebrafish dopaminergic system and quantifiable abnormalities in motor behavior. Chemically-induced brain histamine deficiency causes an identifiable alteration in histaminergic neurons and terminal networks, and a clear change in swimming behavior and long-term memory. Combining the imaging techniques and behavioral methods with zebrafish genetics is likely to help reveal how the modulatory transmitter systems interact to produce important behaviors, and how they are regulated in pathophysiological conditions and diseases.

MPTP and MPP+ target specific aminergic cell populations in larval zebrafish.

Larval zebrafish offers a good model to approach brain disease mechanisms, as structural abnormalities of their small brains can be correlated to quantifiable behavior. In this study, the structural alterations in one diencephalic dopaminergic nucleus induced by 1‐methyl‐4‐phenylpyridinium (MPP+), a toxin inducing Parkinson’s disease in humans, and those found in several neuronal groups after 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), the pretoxin, were associated with decreased swimming speed. Detailed cell counts of dopaminergic groups indicated a transient decline of tyrosine hydroxylase expressing neurons up to about 50% after MPTP. The MPTP effect was partly sensitive to monoamine oxidase inhibitor deprenyl. Detailed analysis of the developing catecholaminergic cell groups suggests that the cell groups emerged at their final positions and no obvious significant migration from the original positions was seen. One 5‐HT neuron group was also affected by MPTP treatment, whereas other groups remained intact, suggesting that the effect is selective. New nomenclature for developing catecholaminergic cell groups corresponding to adult groups is introduced. The diencephalic cell population consisting of groups 5,6 and 11 was sensitive to both MPTP and MPP+ and in this respect resembles mammalian substantia nigra. The results suggest that MPTP and MPP+ induce a transient functional deficit and motility disorder in larval zebrafish.

Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish

Serotonin (or 5‐hydroxytryptamine; 5‐HT) and monoamine oxidase (MAO) are involved in several physiological functions and pathological conditions. We show that the serotonergic system and its development in zebrafish are similar to those of other vertebrates rendering zebrafish a good model to study them. Development of MAO expression followed a similar time course as the 5‐HT system. MAO expression and activity were located in or adjacent to serotonergic nuclei and their targets, especially in the ventral hypothalamus. MAO mRNA was detected in the brain from 24 h post‐fertilization and histochemical enzyme activity from 42 h post‐fertilization. Deprenyl (100 μM) decreased MAO activity 34–74% depending on the age. Inhibition of MAO by deprenyl strongly increased 5‐HT but not dopamine and noradrenaline levels. Deprenyl decreased 5‐HT‐immunoreactivity in serotonergic neurons and induced novel ectopic 5‐HT‐immunoreactivity neurons in the diencephalon in a manner dependent on 5‐HT uptake. Deprenyl administration decreased locomotion, altered vertical positioning and increased heart rate. Treatment with p‐chlorophenylalanine normalized 5‐HT levels and rescued the behavioral alteration, indicating that the symptoms were 5‐HT dependent. These findings suggest that zebrafish MAO resembles mammalian MAO A more than MAO B, metabolizing mainly 5‐HT. Applications of this model of hyperserotonergism include pharmacological and genetic screenings.

Adaptive changes in zebrafish brain in dominant–subordinate behavioral context

Male zebrafish were held in dyadic social stress situation for a period of 5 days, to characterize stress coping styles and to investigate the role of the underlying neuroendocrine mechanisms in establishing dominant-subordinate relationships. A strong consistent dominant-subordinate relationship was formed in ten out of the sixteen pairs of fish (62.5%). Both dominant (DOM) and subordinate (SUB) individuals showed statistically significant higher trunk cortisol concentration than controls. Expression of genes encoding proteins involved in the functioning of the hypothalamus-hypophysis-interrenal axis (corticotropin releasing factor, CRF; glucocorticoid receptor, GR; mineralocorticoid receptor, MR); arginine vasotocin, AVT), in the biosynthesis and catabolism of catecholamines (tyrosine hydroxylase, TH1 and TH2; DOPA decarboxylase, DDC), dopamine β-hydroxylase, DBH; catechol-O-methyl transferase, COMT), in the biosynthesis of histamine (histidine decarboxylase, HDC) and in the general stress response (galanin, GAL; hypocretin/orexin, Hcrt) was examined. The MR/GR ratio was higher in dominant and subordinate fish than in controls (P=0.016). The mRNA levels of TH2 and HDC were up-regulated in DOM, of AVT in SUB, while COMT mRNA levels were down-regulated in both DOM and SUB compared to control fish. In addition, mRNA levels of hypocretin/orexin (Hcrt) were up-regulated in dominant compared to subordinate and control males. There was a statistically significant correlation between mRNA expression levels of TH2, HDC, Hcrt, GR, MR and CRF genes. The obtained results provide new evidences for the use of zebrafish as an animal model to study social stress and allostasis in vertebrates.

The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish

The histaminergic and hypocretin/orexin (hcrt) neurotransmitter systems play crucial roles in alertness/wakefulness in rodents. We elucidated the role of histamine in wakefulness and the interaction of the histamine and hcrt systems in larval zebrafish. Translation inhibition of histidine decarboxylase (hdc) with morpholino oligonucleotides (MOs) led to a behaviorally measurable decline in light‐associated activity, which was partially rescued by hdc mRNA injections and mimicked by histamine receptor H1 (Hrh1) antagonist pyrilamine treatment. Histamine‐immunoreactive fibers targeted the dorsal telencephalon, an area that expresses histamine receptors hrh1 and hrh3 and contains predominantly glutamatergic neurons. Tract tracing with DiI revealed that projections from dorsal telencephalon innervate the hcrt and histaminergic neurons. Translation inhibition of hdc decreased the number of hcrt neurons in a Hrh1‐dependent manner. The reduction was rescued by overexpression of hdc mRNA. hdc mRNA injection alone led to an up‐regulation of hcrt neuron numbers. These results suggest that histamine is essential for the development of a functional and intact hcrt system and that histamine has a bidirectional effect on the development of the hcrt neurons. In summary, our findings provide evidence that these two systems are linked both functionally and developmentally, which may have important implications in sleep disorders and narcolepsy.—Sundvik, M., Kudo, H., Toivonen, P., Rozov, S., Chen, Y.‐C., Panula, P. The histaminergic system regulates wakefulness and orexin/hypocretin neuron development via histamine receptor H1 in zebrafish. FASEB J. 25, 4338–4347 (2011). www.fasebj.org

ARTIFICIAL SELECTION ON RELATIVE BRAIN SIZE REVEALS A POSITIVE GENETIC CORRELATION BETWEEN BRAIN SIZE AND PROACTIVE PERSONALITY IN THE GUPPY

Animal personalities range from individuals that are shy, cautious, and easily stressed (a “reactive” personality type) to individuals that are bold, innovative, and quick to learn novel tasks, but also prone to routine formation (a “proactive” personality type). Although personality differences should have important consequences for fitness, their underlying mechanisms remain poorly understood. Here, we investigated how genetic variation in brain size affects personality. We put selection lines of large‐ and small‐brained guppies (Poecilia reticulata), with known differences in cognitive ability, through three standard personality assays. First, we found that large‐brained animals were faster to habituate to, and more exploratory in, open field tests. Large‐brained females were also bolder. Second, large‐brained animals excreted less cortisol in a stressful situation (confinement). Third, large‐brained animals were slower to feed from a novel food source, which we interpret as being caused by reduced behavioral flexibility rather than lack of innovation in the large‐brained lines. Overall, the results point toward a more proactive personality type in large‐brained animals. Thus, this study provides the first experimental evidence linking brain size and personality, an interaction that may affect important fitness‐related aspects of ecology such as dispersal and niche exploration.

Acute ethanol treatment upregulates th1, th2, and hdc in larval zebrafish in stable networks

Earlier studies in zebrafish have revealed that acutely given ethanol has a stimulatory effect on locomotion in fish larvae but the mechanism of this effect has not been revealed. We studied the effects of ethanol concentrations between 0.75 and 3.00% on 7-day-old larval zebrafish (Danio rerio) of the Turku strain. At 0.75-3% concentrations ethanol increased swimming speed during the first minute. At 3% the swimming speed decreased rapidly after the first minute, whereas at 0.75 and 1.5% a prolonged increase in swimming speed was seen. At the highest ethanol concentration dopamine levels decreased significantly after a 10-min treatment. We found that ethanol upregulates key genes involved in the biosynthesis of histamine (hdc) and dopamine (th1 and th2) following a short 10-min ethanol treatment, measured by qPCR. Using in situ hybridization and immunohistochemistry, we further discovered that the morphology of the histaminergic and dopaminergic neurons and networks in the larval zebrafish brain was unaffected by both the 10-min and a longer 30-min treatment. The results suggest that acute ethanol rapidly decreases dopamine levels, and activates both forms or th to replenish the dopamine stores within 30 min. The dynamic changes in histaminergic and dopaminergic system enzymes occurred in the same cells which normally express the transcripts. As both dopamine and histamine are known to be involved in the behavioral effects of ethanol and locomotor stimulation, these results suggest that rapid adaptations of these networks are associated with altered locomotor activity.

Developmental roles of brain histamine

Histamine appears early during brain development, has been shown to regulate fetal and adult brain-derived stem cells in a receptor type-dependent manner, and has widespread actions on systems involved in arousal and movement. Developmental studies in both rodents and zebrafish have elucidated the spatiotemporal patterning of the histaminergic system and, in zebrafish, have revealed the mechanisms whereby histamine regulates the number of hypocretin/orexin (hcrt) neurons, which in turn may regulate the number of histaminergic cells. Recent demonstrations of increased numbers of histaminergic neurons in patients with narcolepsy highlight the importance, for our understanding of both normal and pathological brain function, of understanding these interactions. Here, we review recent research into the developmental roles of histamine and suggest key areas for future research.

Effect of Ionic Liquids on Zebrafish (Danio rerio) Viability, Behavior, and Histology; Correlation between Toxicity and Ionic Liquid Aggregation

The effect of 11 common amidinium, imidazolium, and phosphonium based ionic liquids (ILs) on zebrafish (Danio rerio) and Chinese hamster ovary cells (CHO) was investigated with specific emphasis on the effect of anion and cation chain length and aggregation of phosphonium based ILs. Viability and behavioral alteration in the locomotor activity and place preference, after IL treatment of 5 days postfertilization larvae, was recorded. Behavior and histological damage evaluation was performed for adult fish in order to get insight into the long-term effects of two potential biomass-dissolving ILs, [DBNH][OAc] and [P4441][OAc]. To get an understanding of how IL aggregation is linked to the toxicity of ILs, median effective concentrations (EC50) and critical micelle concentrations (CMC) were determined. The long-chain ILs were significantly more toxic than the short-chain ones, and the anion chain length was shown to be less significant than the cation chain length when assessing the impact of ILs on the viability of the organisms. Furthermore, most of the ILs were as monomers when the EC50 was reached. In addition, the ILs used in the long-term tests showed no significant effect on the zebrafish behavior, breeding, or histology, within the used concentration range.